Download Chemical genetic approaches for the elucidation of

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Chemical genetic approaches for the elucidation of signaling
pathways
Peter J Alaimo*, Michael A Shogren-Knaak* and Kevan M Shokat*†
New chemical methods that use small molecules to perturb
cellular function in ways analogous to genetics have recently
been developed. These approaches include both synthetic
methods for discovering small molecules capable of acting like
genetic mutations, and techniques that combine the
advantages of genetics and chemistry to optimize the potency
and specificity of small-molecule inhibitors. Both approaches
have been used to study protein function in vivo and have
provided insights into complex signaling cascades.
Addresses
*Department of Cellular and Molecular Pharmacology, University of
California, San Francisco, CA 94143-0450, USA
† Department of Chemistry, University of California, Berkeley,
CA 94720, USA; e-mail: [email protected]
Current Opinion in Chemical Biology 2001, 5:360–367
1367-5931/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviation
1-NM-PP1
1-naphthylmethylpyrazolo[3,5-d]pyrimidine
Introduction
Renewed appreciation for the power of small organic molecules to address questions in cell biology has fueled an
explosion of interest in chemical biology. The idea that
small (MW < 700 Da) drug-like molecules can perturb the
function of specific proteins is a central tenet of pharmacology. This is further bolstered by the fact that many
cellular functions are carried out by small molecules (e.g.
ATP, neurotransmitters, steroid hormones, prostaglandins
and phosphoinositides). Using small molecules to perturb
protein function is particularly useful because the effects
of drugs are:
1. Rapid, potentially diffusion-limited.
2. Often reversible because of metabolism/clearing.
3. Tunable, enabling graded phenotypes by varying
concentration.
4. Conditional, because they can be introduced at any
point in development.
Despite these advantages, the use of small molecules to
probe cellular signaling has lagged behind that of genetic
or biochemical methods. Two types of genetic experiments have provided a wealth of information about cellular
processes. When identification of new components of a
pathway are desired, forward genetics is used. This
involves generating large numbers of mutants, screening to
identify those with either gain- or loss-of-function phenotypes in a process of interest, and then identifying the
mutations in the specific genes that underlie the phenotype. In order to study the function of one component,
reverse genetics is used. Mutations are targeted to a particular protein and the role of the protein is inferred from the
phenotype of the resulting mutant.
The advantages of genetics are that it is both highly specific (a single nucleotide change in 3 billion base pairs
[bps]) and highly portable (any organism can, in principle,
be genetically modified). In contrast, it is often difficult to
identify a small molecule that binds specifically to a single
enzyme active site out of up to 30,000 proteins, some of
which have highly homologous active sites. Additionally,
unlike genetic systems, the synthesis of each small molecule presents unique challenges and is not sufficiently
general to systematically inactivate every gene product in
an organism.
Nonetheless, despite the important advantages of high
specificity and portability there can be problems with traditional genetics. For example, when gene knock-outs are
lethal, further study of the mutant organism is complicated.
Also, most genetic mutations are not conditional; they cannot be turned on or off at will. Although conditional
mutations can be introduced through the use of an inducible
promoter, generation of the mutated protein occurs over
hours to days. Conditional mutations can also be found that
are rapidly manifested by an external stress, such as heat
(TS alleles), but this stress can often have unwanted sideeffects such as induction of heat stress proteins. Thirdly,
knock-out phenotypes of non-essential genes can often be
masked by functional compensation by related genes during
development by the organism. Chemical genetic strategies
using small molecules that act as mutations would complement traditional genetic studies by providing a general
means to rapidly and conditionally inactivate proteins.
The key question becomes, how can we identify specific
chemical inhibitors of every gene product in the yeast,
worm, fly, mouse and human proteomes? The ideal drug is
one that shows perfect target-specific behavior and can be
given to an animal to inactivate its target almost instantaneously. In this review, we highlight two complementary
approaches to generate and identify such molecules, and
describe ways in which these molecules have been used to
study biological processes.
Chemistry as a genetics-like tool
To use small molecules as biological probes, both high
affinity and specificity are necessary. One approach is to
Chemical genetic approaches for the elucidation of signaling pathways Alaimo, Shogren-Knaak and Shokat
find small molecules that are capable of activating or inactivating gene function by direct interaction with the gene
product [1,2]. Historically, natural products have been used
to accomplish this task. To expand the scope of inhibitable
proteins, efforts have focused on generating libraries of
molecules to be screened.
Several recent examples highlight some of the approaches
that are being explored. Since the advent of small-molecule combinatorial synthesis [3–6], countless libraries of
biologically interesting molecules have been synthesized
[7,8]. A subset of these have been based on structural
motifs (scaffolds) that are capable of binding to a variety of
protein targets with high affinity [9]. These structures tend
to be rigid polycyclic heteroatomic systems that are capable of orienting substituents in three-dimensional space.
In a successful recent example, Schultz and co-workers
[10,11] reported the synthesis and application of a focused
library of 2,6,9-trisubstituted purines (Figure 1) in an effort
to identify selective kinase inhibitors. The purine scaffold
was chosen for its ubiquitous appearance in biologically
important molecules. Through iterative chemical synthesis
and screening in vitro, several inhibitors with low nanomolar affinity and some degree of specificity [12] for human
CDK2–cyclin A kinase complex and Saccharomyces cerevisiae Cdc28p were identified. This library has also found use
in other studies [13]. Similarly, syntheses of libraries that
incorporate scaffolds such as benzopyran [14–16], benzodiazepine [17], biphenyl [18], and dihydropyridine [19] have
appeared in the literature [7,8].
As an alternative to privileged structure library synthesis,
diversity-oriented organic synthesis is being investigated.
This approach exploits reactions, such as the Ugi reaction,
that generate a high degree of structural complexity. By
utilizing a range of different building blocks and generating intermediates that can undergo a host of reactions, a
high degree of molecular diversity is introduced [20,21•].
While combinatorial approaches aimed at finding inhibitors
for a particular protein target have been most effective
when structural or mechanistic information about that target has been used in the design process [5,6], recently,
several methods have been described that are useful even
in the absence of such information. One such strategy,
developed by Ellman and co-workers [22•], is to use the target itself to guide the synthesis of the library. In this
method (Figure 2a), a library of monomeric low-affinity ligands is first screened at high concentration. These
monomers are then crosslinked and re-screened to find
more potent bivalent inhibitors. The method was validated
by the discovery of a potent (IC50 = 64 nM) subtype-specific inhibitor of c-Src (>75-fold selectivity over closely related
kinases Fyn, Lyn and Lck). The advantage of this method
is that it allows a high-affinity ligand to be assembled from
its low-affinity components; however, weak ligand–protein
interactions can be difficult to detect. To overcome this
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Figure 1
Cl
R1
1
HN
N
N
R3
R
N
=
R2 =
N
R2
2,6,9-Trisubstituted purine
R3 =
H
N
OH
Current Opinion in Chemical Biology
Chemical structure of the 2,6,9-trisubstituted purine scaffold. The
tightest-binding inhibitor is shown, for which the IC50 against
cdc2–cyclin B is 4 nM.
problem, Wells and co-workers [23] have developed a
method for stabilizing the interaction between weakly
binding molecules and their target by reversible disulfide
bond formation using an endogenous (or mutagenically
introduced) cysteine residue (Figure 2b).
With a small-molecule library in hand, it becomes possible
to perform forward- or reverse-style chemical genetic
experiments, complementary techniques directed toward
different goals. In reverse chemical genetics, the goal is to
determine the biological function of a protein using a small
molecule to inhibit its activity in vivo. To accomplish this,
first the target is chosen, then the chemical library is
screened for potent and selective inhibitors, then the
inhibitors are used to elicit a phenotype. Alternatively, in
forward genetics the goal is to find proteins that are sensitive to small molecule inhibition. This is accomplished by
screening diverse libraries of molecules, identifying those
that elicit an interesting phenotypic response, and identifying the protein target.
To date, small-molecule libraries have been used primarily
in the drug-discovery process, not for dissecting complex
cell signaling pathways. The lead compounds that have
been discovered could be powerful tools for performing
reverse chemical genetics, thus complementing the available natural products that have proved indispensable for
studying cell biology. However, it remains an open question as to whether molecules generated by these methods
will have the required specificity to be useful as biological
probes. A study of the specificity of 28 commercially available, supposedly specific, protein kinase inhibitors against
approximately 30 kinases revealed that all but two drugs
had more than one protein target in vitro [24•]. Admittedly,
kinases have a high degree of homology in their active
sites, and other protein targets could be less problematic.
An important example of the use of chemical libraries to
perform forward chemical genetics comes from the
Schreiber and Mitchison labs [25•]. To find novel
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Figure 2
(a)
X
X
X
X
S
S
X
SR
SR
(b)
Monomer library
SR
SR
S
S
SR
S
Disulfide library
SH
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binding
Protein
target
X
Protein
target
Screen for
binding
Protein
target
X
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target
Protein
target
Methods for identifying small-molecule ligands
based on target-guided self-assembly.
(a) Monomers showing low affinity for their
protein targets can be identified by performing
screens at high concentrations. Optimization
of monomeric lead compounds has been
achieved by crosslinking the ligands and
re-screening to identify highly specific dimeric
inhibitors. (b) Identification of weakly binding
leads has also been achieved using reversible
tethering methods.
S S
Protein
target
Identify
ligand
Identify
ligand
SH
X
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Lead ligands
Lead ligands
Crosslink
ligands
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Protein
target
Rescreen
and identify
optimized
ligands
Optimized ligand
Current Opinion in Chemical Biology
inhibitable proteins in mitosis they devised a screen to
distinguish changes in mitotic spindle formation from
changes in general tubulin polymerization. Small-molecule
inhibitors of mitosis are potential anti-cancer drugs and to
date all known inhibitors of the mitotic machinery, including Taxol and epothilone, target the same protein, tubulin.
Their screen identified monastrol, a molecule that inhibits
normal mitotic spindle formation (Figure 3a,c), but does
not affect normal tubulin function (Figure 3b,d). By correlating this with a known mutant phenotype, and testing
inhibition in vitro, they were able to show that the molecular motor protein Eg5 is a target of monastrol. Hence, this
Chemical genetic approaches for the elucidation of signaling pathways Alaimo, Shogren-Knaak and Shokat
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Figure 3
Monastrol causes monoastral spindles in
mitotic cells. Immunofluorescence staining
(α-tubulin, green; chromatin, blue) of BS-C-1
cells treated for 4 hours with 0.4% DMSO
(control, a,b) or 68 µM monastrol (c,d). No
difference in distribution of microtubules and
chromatin in interphase cells was observed
(b,d). Monastrol treatment of mitotic cells
replaces the normal bipolar spindle (a) with a
rosette-like microtubule array surrounded by
chromosomes (c). Scale Bars, 5 µm.
Interphase
Mitosis
(a)
(b)
(c)
(d)
OH
– Monastrol
O
HN
S
O
N
H
Me
Me
Monastrol
+ Monastrol
Current Opinion in Chemical Biology
study has provided an important new clinical drug lead, a
new therapeutic target, and a potential tool for probing
cell biology.
The forward chemical genetic approach has now been
extended to the study of whole organisms. Using the
zebrafish Danio rerio, Schreiber and co-workers [26•] have
shown that small molecules from diverse libraries can elicit interesting phenotypes during development. This
example highlights a key challenge for forward chemical
genetics, that is to identify the protein targets of smallmolecule inhibition. For phenotypes that cannot be easily
correlated with a unique mutation, some methods are
available including affinity purification [27], yeast threehybrid systems [28,29], display cloning [30,31], and protein
microarrays [32,33]. A potentially useful genetic solution is
to generate and isolate mutations that abrogate drug-sensitivity. Recently, this idea has been used to identify the
target of a small-molecule plant hormone that had long
eluded identification [34•].
Another complication in performing forward chemical
genetics is that small molecules can have multiple
modes of action and inhibit multiple targets. The
mechanism of vancomycin inhibition is a case that
demonstrates this. Although one mode of action of vancomycin is binding to the D-Ala–D-Ala motif in the
peptidoglycan, recent work by Kahne and co-workers
[35•] demonstrates that a mechanism independent of
peptide-binding can also operate.
Overall, the various methods for generating and using
libraries in both forward and reverse chemical genetics
offer the potential to discover new small molecules that
will be useful for probing biological processes and providing new drugs. We expect that the examples discussed
herein will serve as a foundation for continued discovery in
cell biology.
Chemistry coupled to genetics
A fundamentally different way of using chemistry to study
biology is to combine chemistry with genetics in one
experimental regime [36]. When trying to perturb biological function using small molecules, the central problem is
to find small molecules that interact specifically with a
desired protein target. Generating specific drugs is especially challenging when the protein target shares a high
degree of homology with other proteins in the cell. When
using drugs as biological probes, however, one is not limited to modifying the small molecule. Recombinant DNA
technology enables modulation of protein structure by
introducing point mutations or even entire protein
domains. Thus, it is possible to start with a high affinity
but non-specific inhibitor and confer specificity by sitedirected mutagenesis. Alternatively, one can genetically
introduce protein domains that already possess high affinity and specificity. Pioneering work by Hwang and Miller
[37] demonstrated that by interchanging the hydrogenbonding donor–acceptor groups between substrate and
enzyme, they could convergently engineer a GTPase to
uniquely accept XTP, allowing them to probe a number of
GTPase-dependent processes [38–40]. Elegant realization
of the latter idea has been demonstrated by Schreiber and
co-workers [41], in which grafting of protein domains that
dimerize in a small-molecule-dependent fashion provides
a chemically inducible means of associating target proteins.
Our laboratory has developed a combined chemical genetic method to specifically inhibit protein kinases [42–44].
Protein kinases form a large enzyme family and play a significant role in nearly all signaling pathways [45]. The
active site of protein kinases is well conserved and makes
specific inhibition of a desired kinase challenging [46]. To
generate protein-inhibitor specificity, we use protein design
to engineer a functionally silent yet structurally significant
mutation into the active site of a kinase of interest. In our
case, this mutation is the replacement of a conserved bulky
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Next generation therapeutics
Figure 4
(a)
(b)
WT kinase with
general inhibitor
WT kinase with
modified inhibitor
Protein substrate
Protein substrate
Adenosine P P P
WT
kinase
Adenosine P P P
WT
kinase
Inhibitor
Inhibitor
NH2
Adenosine P P
(e)
N
N
N
N
PPPO
P
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N
HO
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modified inhibitor
(d)
General kinase inhibitor PP1
Mutant kinase with
modified inhibitor
Protein substrate
(f)
Protein substrate
Adenosine P P P
Adenosine P P P
Mutant
kinase
Mutant
kinase
N
OH
ATP
(c)
N
N
O
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NH2
Inhibitor
NH2
NH2
N
N
N
N
N
N
N
N
Adenosine P P
28,000/4.2
IC50 WT/mutant (nM)
1000/1.5
IC50 WT/mutant (nM)
P
Protein substrate
Protein substrate
Specific kinase inhibitors
Current Opinion in Chemical Biology
Kinase-specific inhibition can be achieved by coupling chemistry and
genetics. (a) Wild-type kinases are inhibited by a non-specific
inhibitor. (b) Wild-type kinases are not inhibited by non-specific
inhibitor analogs containing a sterically bulky functional group.
(c) Engineered kinases show normal kinase activity. (d) Engineered
kinases are inhibited by inhibitor analogs that contain a sterically
bulky functional group. (e) Chemical structures of ATP and the
general kinase inhibitor PP1. (f) Chemical structures of specific
kinase inhibitors (structure in red indicates modified group), IC50
WT/mutant (nM). WT, wild type.
residue with glycine or alanine, thus creating a new pocket
in the active site. Importantly, these mutations do not usually affect kinase activity in a significant way (Figure 4c).
Separately, a non-specific inhibitor of the wild-type enzyme
(Figure 4a,e) is chemically modified with substituents that
specifically complement the mutation introduced into the
active site (Figure 4d,f). Importantly, the new inhibitor
analogs are designed to be unable to inhibit any wild-type
kinases via steric clashes (Figure 4b). In a cellular context,
it is difficult to prove perfect specificity. Experiments have
shown that our inhibitors have no off-target effects in vitro;
furthermore, addition of our inhibitors to wild-type
S. cerevisiae has resulted in few transcriptional changes
[47•]. Using this approach, several analogs of a pyrazolo[3,5-d]pyrimidine-based kinase inhibitor (PP1) have been
found that inhibit engineered kinases with nanomolar
IC50 values and without significant inhibition of wild-type
kinases [44,48].
This combined chemical genetic strategy is potentially
general for many kinases, but several requirements must
be met. First, it must be possible to introduce the desired
mutant allele into the organism of interest. Second, the stability and activity of the engineered enzyme must be
unaltered. Third, the inhibitor must be bioavailable. Work
in our labs and in collaboration with others has demonstrated that these requirements can be met in most
circumstances. This technique has been used to probe the
functional role of CaMKIIα in learning and memory
in mice (JZ Tsien, personal communication), v-Src in
Chemical genetic approaches for the elucidation of signaling pathways Alaimo, Shogren-Knaak and Shokat
transformation of 3T3 cells [44], and Cla4 and Cdc28 in
cell-cycle regulation in S. cerevisiae [47•,49•].
An example that highlights the ability of chemical genetics
to complement traditional genetics is the use of this technique to clarify the role of Cdc28 in the cell cycle of
S. cerevisiae. Cdc28 is the primary cyclin-dependent kinase
involved in cell-cycle control. Use of mutants containing a
temperature-sensitive Cdc28 allele generated by traditional
genetics suggested that the most critical role for Cdc28 was
to control the transition from G1 to S phase. Surprisingly,
experiments done in collaboration with David Morgan
(UCSF) showed that inhibitor-sensitive Cdc28 mutants
arrested at the G2/M transition when treated with 1-naphthylmethylpyrazolo[3,5-d]pyrimidine (1-NM-PP1). This
discrepancy was not due to off-target effects of the drug,
because this compound induced no toxicity or cell-cycle
arrest in wild-type yeast. Thus, the difference in cellular
phenotype was the result of a fundamental difference in
how cdc28 function was altered by the two approaches.
The discrepancy between the chemical and temperatureinduced inhibition of cdc28 can be explained from a
structural perspective. Cdc28 functions in two ways, as a
catalyst and as a scaffold for other components of the cellcycle machinery. Typically, temperature-sensitive mutants
unfold at elevated temperature thus resulting in a loss of
both of these functions. ATP-competitive inhibitors, such
as 1-NM-PP1, block only kinase catalytic activity. Thus, in
this case, the chemical method is a more specific probe of
protein function.
Moreover, the observed discrepancy is consistent with
what is known about cdc28 catalytic activity. The kinase
activity of Cdc28 is maximal at the G2/M transition and is
therefore expected to be most sensitive to inhibition at this
stage, consistent with observed G2/M arrest at low doses of
1-NM-PP1. This model predicts that higher inhibitor concentration should result in earlier cell-cycle arrest. This
was also demonstrated. Thus, the ability of this chemical
genetic approach to probe protein function more specifically highlights the advantages of using chemical genetics
as a complement to traditional genetics.
In addition to using combined chemical genetics to inhibit proteins, it is also possible to probe the activity of
enzymes and receptors using modified agonists and substrates. Hence, we have applied our strategy toward
identifying kinase substrates. By synthesizing γ[32P]labeled analogs of ATP that contain a sterically bulky
functional group and using the engineered kinases previously described, we can selectively label the direct
phosphorylation substrates of a kinase of interest [50,51].
Conklin and co-workers [52,53•] have applied a chemical
genetic method to the study of seven-transmembrane Gprotein-coupled receptors. By introducing changes in the
agonist-binding domain they have generated receptors
that are activated by a synthetic agonist, but not the
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endogenous peptide hormone ligand. By providing the
researchers with exclusive control over the activation of
these receptors, influence over a number of physiological
processes including heart rate and cell proliferation has
been demonstrated.
Approaches that combine chemistry and genetics offer a
means of generating small molecules that act rapidly and
reversibly, yet offer the portability and specificity of genetics.
Conclusions and future directions
The intersection between genomics, proteomics, combinatorial organic synthesis, natural-product screening, and
protein design has created an exciting environment for the
development of powerful new tools that have been termed
chemical genetics. To date, our view of biological phenomena has been shaped largely through the use of
genetics. Yet, we know that genetics has limitations. The
use of chemical agents to probe pathways provides information that, when superimposed with genetic and
biochemical data, gives a higher resolution understanding
of biological processes. An ongoing challenge is to identify
biological questions that benefit from the application of
small-molecule approaches. Another is to continue to
develop tools that are sufficiently specific and portable to
address these questions. In meeting these challenges,
chemical genetics offers the potential to augment and even
profoundly expand our understanding of biology.
Acknowledgements
We are grateful to V Denic, HF Luecke, JW Taunton and members of the
Shokat laboratory for helpful discussions and critical comments on this
manuscript. PJA thanks the Susan G Komen Breast Cancer Foundation
(PDF 2000 78) and the American Cancer Society (PF-01-095-01-TBE), and
MSK thanks the National Institutes of Health (AI10611-01) for postdoctoral
fellowships. KMS thanks the National Institutes of Health (CA70331-05
and AI44009-03), GlaxoSmithKline and the Sandler Family Supporting
Foundation for generous financial support.
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Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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